The present invention relates to a novel reagent for the assay of amylase isozyme activity and assay methods using the reagent. More particularly, the present invention relates to a novel reagent for simple assay of amylase isozyme activity, comprising an antibody which inhibits S-type amylase activity or P-type amylase activity but not containing an adjuvant enzyme and assay methods using the reagent.
xcex1-Amylase contained in a body fluid, such as pancreatic juice, urine and the like, is mainly classified into two kinds of isozymes of P-type amylase mostly derived from pancreas and S-type amylase mostly derived from salivary gland. Various diseases are diagnosed by assaying these isozymes. Of these, P-type amylase is said to be useful as a marker of acute pancreatitis, since it has high organ specificity.
Conventionally, an electrophoretic method and a method using a wheat-derived amylase activity inhibitor have been used for amylase isozyme assay. These methods, nevertheless, are defective in that the operation is complicated and time-consuming, P-type amylase activity cannot be assayed directly, and so on. For example, a method using a wheat-derived amylase inhibitor is known, but the isozyme selectivity of the inhibitor is insufficient in this method. As a result, P-type amylase activity cannot be assayed directly so that complicated quantitative calculation becomes necessary, such as depicting a calibration curve and the like. Although an electrophoretic method was known long before that, this method requires complicated operation and skillful interpretation of the results, which have been pointed out as defects.
Meanwhile, various methods utilizing different reactivities of isozymes to a certain substrate or two kinds of substrates having different structures have been disclosed (Japanese Patent Examined Publication No. 24998/1992; Japanese Patent Unexamined Publication No. 98498/1989; Japanese Patent Unexamined Publication No. 193892/1992; Japanese Patent Unexamined Publication No. 229196/1992; Japanese Patent Examined Publication No.52279/1992; and the like). These methods again require complicated reactions that prevent direct assay of P-type amylase activity and necessitate complicated quantitative calculation involving depicting a calibration curve and the like.
A method using ferric ion as an inhibitor has been also disclosed (Japanese Patent Unexamined Publication No. 209600/1986). This method similarly requires complicated quantitative calculation.
In recent years, methods using a monoclonal antibody having a higher isozyme selectivity have been disclosed. A method using an anti-P-type isozyme monoclonal antibody is known Japanese Patent Examined Publication No. 46116/1991), but this method has a defect in that the manipulation is complicated due to an additional step for separating an antigen-antibody complex from others.
In addition, a method is known wherein one or several kinds of monoclonal antibodies is/are used to specifically inhibit S-type isozyme alone, whereby P-type isozyme activity is specifically assayed (Japanese Patent Examined Publication No. 37380/1992 and Japanese Patent Examined Publication No. 2600/1988).
While the substrates for amylase assay to be used for these methods are not particularly limited, it does not necessarily mean that any substrate for amylase assay can be used for these methods. Moreover, since the substrate for amylase activity assay to be combined has superiority or inferiority in performance, which reagent to select is practically critical
The reagent for xcex1-amylase activity assay to be combined with the method using the above-mentioned antibody is often a reagent for general use for an automatic analyzer. Of such reagents, reagents containing various maltooligosaccharides or derivatives thereof as substrate are typically used. For example, (1) maltooligosaccharide having glucose units of 4-7, (2) a derivative (glucose units 4-7) wherein aglycon, which becomes optically assayable upon liberation, has been bound with the reducing terminal of maltooligosaccharide, (3) a derivative (glucose units 4-7) wherein aglycon, which becomes optically assayable upon liberation, has been bound with the reducing terminal of maltooligosaccharide and the 4- and/or 6-position hydroxyl group of non-reducing terminal glucose have/has been modified, and the like may be used.
In the method using the reagent of the aforementioned (1) and (2), the adjuvant enzyme such as xcex1-glucosidase, which has been added to lead the hydrolysate resulting from the amylase reaction into a detectable substance, acts to increase reaction of the blank. The method using the reagent of (3) theoretically enables to obviate the action of xcex1-glucosidase and, therefore, is more preferable than the above methods which use a reagent of (1) and (2). In practice, however, a by-product, wherein the non-reducing terminal is not modified, is somewhat contained and a slight increase in the blank value is inevitable. The use of adjuvant enzyme also poses a problem of high cost. These problems similarly occur when a reagent and an antibody are combined for assaying isozyme.
Thus, (4) a method using 2chloro-4-nitrophenylmaltotrioside, which obviates the need of adjuvant enzyme, as a substrate has been developed (Japanese Patent Unexamined Publication No. 183595/1988), which circumvents the aforementioned problems in xcex1-amylase activity assay. This method, nevertheless, has low sensitivity, and requires addition of thiocyanate or azide at a high concentration as an amylase activator. When this method and an antibody are combined to assay amylase isozyme (Clin. Chim. Acta., No. 259, pages 147-160, 1997), however, the high concentration activator prevents sufficient S-type amylase inhibitory activity. To avoid this, the activator content needs to be lowered to a certain level, which degrades the sensitivity of the method. Moreover, the addition of azide at a high concentration poses problems of possible toxicity and creation of an explosive compound upon bonding with a heavy metal.
The present invention therefore aims at providing a reagent for easy and precise assay of amylase isozyme activity, which contains a low concentration activator and an antibody inhibiting S-type amylase activity or P-type amylase activity but does not contain adjuvant enzyme, and assay methods using this reagent.
According to the present invention, there has been provided the use of 2-chloro-4-nitrophenyl 4-O-xcex2-D-galactopyranosylmatoside as a substrate for amylase activity assay.
That is, the present invention provides a reagent for assaying amylase isozyme activity, which characteristically contains 2-chloro4-nitrophenyl 4-O-xcex2-D-galactopyranosylmaltoside, an antibody inhibiting S-type amylase activity or P-type amylase activity, and an amylase activator.
The present invention also provides a method for assaying amylase isozyme activity, which comprises the steps of:
(a) bringing a reagent for amylase isozyme activity assay, which contains 2-chloro-4-nitrophenyl 4O-xcex2-D-galactopyranosylmaltoside, an antibody having S-type amylase activity or P-type amylase activity-inhibitory activity, and an amylase activator, into contact with a sample containing P-type amylase and S-type amylase to allow reaction of 2-chloro-4-nitrophenyl 4-O-xcex2-D-galactopyranosylmaltoside and the P-type amylase or S-type amylase, and
(b) assaying a content of liberated 2-chloro-4-nitrophenol.
Moreover, the present invention provides a method for assaying amylase isozyme activity, which comprises the steps of
(a) bringing a reagent for amylase isozyme activity assay, which contains 2-chloro-4-nitrophenyl 4-O-xcex2-D-galactopyranosylmaltoside and an amylase activator, into contact with a sample containing P-type amylase and S-type amylase to allow reaction of 2-chloro-4nitrophenyl 4-O-xcex2-D-galactopyranosylmaltoside and the P-type amylase and S-type amylase,
(b) assaying a content (A) of liberated 2-chloro-4-nitrophenol,
(c) bringing a reagent for amylase isozyme activity assay, which contains 2-chloro-4-nitrophenyl 4-O-xcex2-D-galactopyranosylmaltoside, an antibody having S-type amylase activity or P-type amylase activity-inhibitory activity, and an amylase activator, into contact with a sample containing P-type amylase and S-type amylase to allow reaction of 2-chloro-4-nitrophenyl 4-O-xcex2D-galactopyranosylmaltoside and the P-type amylase or S-type amylase,
(d) assaying a content (B) of liberated 2-chloro-4-nitrophenol, and
(e) subtracting the (B) from the (A).